Effect of Low-Intensity Pulsed Ultrasound After Autologous Adipose-Derived Stromal Cell Transplantation for Bone-Tendon Healing in a Rabbit Model

Background: Low-intensity pulsed ultrasound (LIPUS), as a safe biophysiotherapy, can enhance bone-tendon (B-T) healing in vivo and induce osteogenic or chondrogenic differentiation of mesenchymal stromal cells in vitro. This study aimed to determine whether LIPUS can improve the efficacy of transplanted mesenchymal stromal cells on B-T healing. Hypothesis: LIPUS can induce lineage-specific differentiation of transplanted adipose-derived stromal cells (ASCs) at the B-T healing site, thus resulting in superior healing quality when compared with LIPUS or ASCs alone. Study Design: Controlled laboratory study. Methods: A total of 112 mature rabbits with partial patellectomy in the hindlimb were randomly assigned into mock sonication without ASCs (control), ultrasonication without ASCs (LIPUS), mock sonication with ASCs (ASCs), and ultrasonication with ASCs (LIPUS + ASCs). The treatment time of the mock sonication or ultrasonication was 20 minutes per day. Autologous ASCs were transplanted to the healing site by fibrin glue during the operation, and LIPUS was delivered daily starting at postoperative day 3 until euthanasia. The patella–patellar tendon junctions were postoperatively harvested at 8 and 16 weeks for radiological, histological, and mechanical evaluations. Additionally, 9 animals were used for ASC tracking with mCherry protein. Results: Radiologically, there was more new bone formation and remodeling in the LIPUS + ASCs group as compared with the other groups. Synchrotron radiation micro–computed tomography showed that the LIPUS + ASCs group significantly increased bone volume fraction, trabecular thickness, and trabecular number at the healing site as compared with the other groups at postoperative 8 weeks ( P < .05 for all). Histologically, immunohistochemical staining confirmed that the transplanted mCherry-ASCs can differentiate into osteoblasts and fibrochondrocytic-like cells. Meanwhile, as compared with the other groups, the LIPUS + ASCs group showed more formation and maturity of the fibrocartilage layer and new bone at postoperative weeks 8 and 16 ( P < .05 for all). Biomechanically, the LIPUS + ASCs group showed significantly higher failure load and stiffness versus the other groups at postoperative weeks 8 and 16 ( P < .05 for all). Conclusion: Autologous ASC transplantation stimulated with LIPUS can result in superior B-T healing quality when compared with LIPUS or ASCs alone. Clinical Relevance: This study demonstrates the effectiveness of using ASC transplantation stimulated with LIPUS for B-T healing and provides a foundation for future clinical studies.